The non-pathogenic yeast Candida bombicola and other yeast species such as Candida apicola, Candida batistae, Rhodotorula bogoriensis and Wickerhamiella domericqiae are known for their sophorolipid production during stationary phase (Spencer et al., 1970, Gorin et al., 1961, Tulloch et al., 1968, EP0837140A1, U.S. Pat. No. 6,433,152, U.S. Pat. No. 4,215,213). C. bombicola and others are oleaginous yeast species, i.e. they can utilize oleaginous substrates such as alkanes and oils as carbon source, and can handle those substrates in relatively high concentrations. Moreover, C. bombicola can produce sophorolipids in high amounts (over 400 g/L), which are excreted in the fermentation medium.
Candida bombicola ATCC 22214 is already applied commercially for the production of sophorolipids. These glycolipid biosurfactants are constituted of a sophorose head group (2-O-β-D-glucopyranosyl-β-D-glucopyranose) from which the anomeric C-atom is attached to an (ω) or (ω-1) C16 or C18 hydroxylated fatty acid. They occur either as open-ring structures (acid form) or as lactones with an intra-esterification between the fatty acid carboxyl group and the 4″, 6′ or 6″ carbon atom of the sophorose head group. In addition, acetyl groups can be attached at the 6′ and/or 6″ positions (Asmer et al., 1988).
In a typical Candida bombicola fermentation with e.g. rapeseed oil as a hydrophobic carbon source, sophorolipids are present as a complex mixture of structurally related molecules with the mono- and di-acetylated lactonic sophorolipids being the most important.
While many research has focused on fermentation conditions to optimize sophorolipid production by C. bombicola (Daniel et al., 1998a, Daniel et al., 1998b, Casas et al., 1999, Cavalero et al., 2003, Kim et al., 2009), and sophorolipids have served as substrates for (chemo)-enzymatic modifications (Bisht et al., 1999, Hu et al., 2003, Rau et al., 1999), the biochemical pathway of these economically important bioproducts remains unclear and there is no information available about the genes involved (Ochsner et al., 1994a, Ochsner et al., 1994b). This lack of information hampers implementation of modern techniques such as metabolic engineering to increase sophorolipid yields. The only data about enzymes involved in sophorolipid production by C. bombicola suggest the involvement of a cytochrome P450 monooxygenase from the CYP52 family (Van Bogaert et al., 2009). Data about enzymes in other yeasts are limited to protein experiments with C. bogoriensis lysates and date from early 1970's and 1980's (Esders et al., 1972, Bucholtz et al., 1976, Breithaupt et al., 1982). Apart from an acetyltransferase, it was assumed that two glucosyltransferases were involved in the stepwise production of sophorolipid by this organism, though separation of the two activities remained unsuccessful (Breithaupt et al., 1982).
Besides the fact that the biochemical pathway of the production of sophorolipids is largely unknown, C. bombicola will always produce sophorolipids no matter which carbon source is applied. Hence, the sophorolipids will always form a mixture with other compounds of potential interest and the biosynthetic pathway will compete for the use of substrates. Consequently, it is hardly impossible to combine the (recombinant) production of a compound of interest with the sophorolipid synthesis without substantial loss of efficiency and without additional purification costs. The option to only produce the compound of interest during the exponential growth phase when no or low amounts of sophorolipids are produced, will not only result in lower yields due to the lower amount of available biomass and shorter production time, but will also often result in a low tolerance of the growing cells towards the oleaginous substrates used. These problems are omitted when strains that lack this sophorolipid production are used. However, Ito and Inoue (Ito et al., 1982; Inoue et al., 1982) demonstrated that sophorolipids stimulate the growth on oleaginous substrates whereas a number of synthetic non-ionic surfactants have no effect. They further state that sophorolipids act as specific growth stimulating factors and are needed to emulsify the insoluble oleaginous substrates. Therefore, strains unable to produce sophorolipids show inferior growth as compared to the wild type and are unable to handle oleaginous substrates.
Taken together, it is clear that sophorolipid-producing yeast species might be very useful to produce, in addition to sophorolipids, other numerous useful compounds such as recombinant proteins, glycolipids, polyhydroxyalkanoates, sophorose, rhamnose, special fatty acids, squaleen, organic acids, hydrophobic compounds and oleagenious compounds. However, a lack of understanding of the underlying biochemical pathway of sophorolipid synthesis and the problem of combining the production of a useful compound of interest with the sophorolipid synthesis without substantial loss of efficiency and without additional purification costs seriously hamper the usage of these yeast species.
Description of Tables:    Table 1 Primers used for knocking-out the C. bombicola CYP52M1 gene. All primers were obtained from Sigma Genosys.    Table 2 Primers used for isolation of the UGTA1 gene and construction of the knock-out cassette. All primers were obtained from Sigma Genosys.    Table 3 Ten best homology scores for the translated UGTA1 sequence    Table 4 Primers used for isolation of the UGTB1 gene and construction of the knock-out cassette. All primers were obtained from Sigma Genosys.    Table 5 Ten best homology scores for the translated UGTB1 sequence    Table 6 Primers used for creation of the GFP expression cassette. All primers were obtained from Sigma Genosys. Bold characters represent non-binding extensions.    Table 7 Primers used for creating the amylase expression cassette and control of amylase transformants.    Table 8 Primers used for creating the PHA expression cassette. All primers were obtained from Sigma Genosys.    Table 9 Primers used for creation of the UGT1 and CepB expression cassettes. All primers were obtained from Sigma Genosys.